The proposed research aims at a precise molecular understanding of how certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum membrane in mammalian cells. We will identify the cellular constituents of the protein targeting and translocation machinery that are essential, and ultimately also those that are modulatory, to gain a detailed understanding of the mechanism of targeting and translocation and the role which individual molecules play in this fundamentally important process. Specifically, i) we will determine the molecular details of signal recognition particle (SRP) - signal sequence interactions and SRP-ribosome interactions and ask how these interactions are regulated. ii) We will determine the individual contribution of each of the three GTPase domains contained in the SRP and the SRP receptor during protein targeting to the endoplasmic reticulum membrane and the assembly of the protein translocation site. We will design assays to identify other molecules that affect the binding and/or hydrolysis of GTP by individual GTPase domains. iii) We will determine the minimal set of ER proteins that is required for nascent chain targeting, nascent chain insertion and nascent chain translocation and try to decipher the mechanistic role that these protein play in the process. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.